Analytica Chimica Acta, 162 (1984) 333--338 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

DETERMINATION OF ORGANOPHOSPHORUS COMPOUNDS BY DYE-ASSISTED CHROMATOGRAPHY

ANDRES TRUJILLO, T. GNANASAMBANDAN and HENRY FREISER*

Strategic Metals Recovery Research Facility, Department of Chemistry, University of Arizona, Tucson, AZ 85721 (U.S.A.)

(Received 23rd January 1984)

SUMMARY

The application of dye-assisted reversed-phase liquid chromatography for the deter- mination of several organophosphorus compounds is demonstrated for mobile phases containing 10 -4 M Brilliant Green in a mixed solvent of water and methanol, acetonitri-le and 1,4-dioxane. This method, based on the interactions between a dye and analyte molecules, offers good detection limits for compounds which have weak chromophores. Detection limits are in the submicrogram range for aromatic pesticides, and in the low microgram range for non-chomophoric aliphatic compounds.

Organophosphorus compounds are commonly used in agriculture as insec- ticides [1], and in industry as extractants for the recovery of metals [2]. As insecticides, these compounds have the advantage of having low toxicity toward mammals and also of being degraded easily into less toxic compounds.

The preferred method for the separation, quantitation, and identification of organic phosphorus compounds has been gas chromatography (g.c.) with photometric or with mass spectrometric detection [3, 4]. A problem asso- ciated with the g.c. method is the thermal decomposition of the sample. This problem can be overcome by using other chromatographic techniques such as thin-layer, ion-exchange, and high-performance liquid chromatography (h.p.l.c.) which use milder conditions for separation [3]. Of the previous chromatographic techniques, h.p.l.c, seems to be the most promising for the rapid separation and quantitation of neutral organic phosphorus compounds because of its high resolving power and the capacity to preconcentrate and cleanup samples using practically the same hardware [ 5, 6]. A serious draw- back of h.p.l.c, with photometric detection is the lack of sensitivity for com- pounds having weak chromophores [ 3].

Efforts to improve photometric detection of non-absorbing ionic com- pounds has been successful to a considerable extent using ion-pair chromato- graphy [7--9]. This technique, however, can be applied only for the more easily ionizable organophosphorus acids and amines. This study deals with the photometric detection of neutral nonabsorbing aliphatic phosphorus compounds as well as the enhanced detection of neutral aromatic phosphorus

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compounds using reversed-phase h.p.l.c, with a dye-containing mobile phase. The method consists in the equilibration of a reversed-phase column with a mobile phase containing a dye. Once equilibration has been achieved, the sample is injected on the column and the same mobile phase is used for elution. Detect ion of neutral nonchromophoric analytes is possible because of the interaction between the analyte and the dye molecules which cause an increase in the molar absorptivity of the dye at several wavelengths. The interaction between the analyte and the dye is fairly general, and several families of neutral compounds including alcohols, ketones, esters and nitriles, can also be detected with this method [10, 11] .

EXPERIMENTAL

Apparatus Two chromatographic systems were used in this study. The first consisted

of an Altex pump (model 110 A), a 10-pl loop rotary valve injector (Spectra- Physics, SP-419-0410), and a Whatman Partisfl ODS-2 (10 ~m, 25 cm X 0.46 cm) column. The second chromatographic system consisted of a dual- pump Spectra-Physics liquid chromatograph (SP-3500B) equipped with another 10-pl loop rotary-valve injector, and a slurry-packed Spherisorb $5- ODS-2 column (5 pro, 25 cm X 0.46 cm).

The detectors used were a Spectra-Physics SP-8200 u.v./visible selectable wavelength detector equipped with a 254-nm or 436-nm filter, and a refrac- tive index detec tor (Showa Denko KK Shodex RI Model SE-11).

Mobile phase. The mobile phases contained 0.10 × 10 -3 M Brilliant Green and various concentrations of water and organic solvent. Cleaning and equil- ibration of the columns were as described previously [ 10] . The flow rate was maintained at 1.0 ml min -1. The temperature was maintained at 30°C. The volume of the mobile phase was taken as the breakthrough volume.

Chemicals and stock solutions The organophosphorus compounds used in this s tudy were obtained from

several sources, and were used without further purification. The presence of trace impurities in these compounds was established by inspection of chomato- grams obtained for each compound studied and were found to be noninter- feting (i.e., peaks are small and completely resolved from the main compo- nent). Deionized water was degassed under vacuum at 50 ° C. Reagent-grade methanol and acetonitrile were used as received. 1,4-Dioxane was refluxed over sodium metal for three days and then distilled from glass before use. Brilliant Green hydrogen sulfate (94% pure; Eastman) was used as received.

Stock solutions of organophosphorus compounds were prepared by care- fully weighing the compound in a volumetric flask and then diluting it with dioxane or, in some cases, with mobile phase. Standards prepared in aqueous solutions were used within one day. Calibration graphs were prepared by replicate injection of 10 gl of standard. Five standards were used for every compound.

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RESULTS AND DISCUSSION

The dye used here was the hydrogensulphate salt of Brilliant Green. This dye is strongly adsorbed by the reversed-phase column and exhibits high sensitivity for the detection of nonchromophoric neutral substances. The selection of Brilliant Green was based on the requirements of water solu- bility, sensitivity, and stability towards decomposition. Other dyes meeting the above requirements are Methylene Blue, Nile Blue A, and Acridine Orange.

[ ~ C / ~ N(CH2CH 3)2

HSO~

N(CH 2CH3) 2 +

The wavelength of detection used was not always that of maximum sensi- tivity. For convenience, 254 or 436 nm was selected when the Spectra- Physics detector was used. The wavelength of maximum sensitivity can be obtained by comparing the spectra of the mobile phase in the absence and presence of analyte [12, 13].

The analytes used in this study were members of the phosphates, phos- phites, phosphonates, phosphorothionates, and phosphorodithionates family. Aliphatic as well as aromatic compounds were included in order to compare the detection sensitivity for both nonabsorbing and strongly absorbing com- pounds. Because the compounds used were widely different in molecular weight and functionalities, it was necessary to study them for different mobile-phase compositions. Capacity factors for several organic phosphorus compounds including pesticides are listed in Tables 1--3. Table 1 shows the capacity factors for several compounds of low molecular weight eluted with methanol or dioxane in the mobile phase. Elution with dioxane-water gives good results for the separation, but its utility for the elution of compounds of higher molecular weight (e.g., aromatics) was limited by the increased back-pressure produced by this highly viscous solvent. Instead of continuing working with dioxane in the mobile phase, methanol and acetonitrile-water mixture were considered.

Capacity factors for compounds of low molecular weight obtained with different methanol-water mobile phases are shown in Table 1. The capacity factors obtained with methanol as organic modifier are considerably higher than those obtained with 10% dioxane-water. Thus, the solvent strength of the former is expected to be ineffective for the elution of the phosphorus compounds with higher molecular weight. For the elution of the more hydro- phobic compounds, acetonitrile-water mixtures were found to be appropriate.

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TABLE 1

Capacity factors (k') obtained with methanol or dioxane in the mobile phase a

Compound Methanol (%)

10 15 30 40

Dioxane (%) 10

Dimethyl methyl- 1.8 1.6 0.7 -- 0.8 phosphonate Diethyl phosphite 5.0 3.9 1.8 1.4 2.5 (DEPI) Hexamethyl- 10.4 7.9 4.5 2.8 3.5 phosphoramide (HMP) Diethyl ethyl- 13.0 9.4 4.5 3.5 6.9 phosphonate (DEEPO)

aMobile phase: 0.10 mM Brilliant Green and x % methanol or dioxane (v/v). Partisil ODS- 2 column with detection at 254 nm.

TABLE 2

Capacity factors obtained with 50% acetonitrile

Compound k 'a Compound k 'a Compound k 'a

Methyl parathion 4.5 Dibutyl butyl- 9.6 EPN 17.6 Triphenyl phosphate 9.2 phosphonate Tri-(o-tolyl) 28.9 Ethyl parathion 9.5 Fenthion 9.7 phosphate Tribu tyl phosphate 9.5 Coumaphos 11.5 Ethion 35.0 Diazinon 9.6 Phorate 12.4 Carbophenothion 40.5

aMobile phase was 0.10 mM dye, 2.0 mM ammonium phosphate, and 50% acetonitrile- water. Spherisorb S5-ODS-2 column.

In Table 2, capacity factors for some phosphorus compounds having molecular weights greater than 200 are shown. Elution with 50% acetonitrile- water was not effective for the separation of all the compounds shown in Table 2. Thus, although separation of methyl and ethyl parathion was pos- sible, resolution of dibutyl butylphosphonate from its analog, tributyl phos- phate, was impossible under the present conditions.

The effect of the dye on the retention of the organic phosphorus com- pounds was investigated by comparing the capacity factors of the compounds eluted in the presence (k') and absence (K") of dye. From the results obtained (Table 3), a linear relationship having a correlation coefficient (r) of 0.9998 and regression equation: k' = (1.02 + 0.01) K" + (0.82 -+ 0.04), was obtained. Thus, the net effect of the dye on retention is to increase the capacity factor by 0.82 units. Analogous relationships have been obtained before for aliphatic alcohols [10]. The increase in capacity factors caused by the dye, both

TABLE 3

Comparison of capacity factors in presence and absence of dye a

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Compound K" (0.0 raM) a k' (0.10 raM) b

Dibutyl phosphite 0.5 1.3 Dibutyl butylphosphonate 2.3 3.2 Tributyl phosphate 2.3 3.2 Tri-(o-tolyl) phosphate 4.7 5.6

aMobile phase was 75% (v/v) methanol-water; Partisil ODS-2 column; refractive index detector, bSystem as for preceding column but with 0.10 mM dye added, and u.v.-visible detection at 436 rim.

adsorbed in the column and in the mobile phase, may be ascribed to the increased interaction of the analyte and the adsorbed dye and the possible formation of a dye-analyte adduct having a larger partition coefficient favor- ing retention, as proposed earlier [ 10] . Further work is necessary to elucidate the actual mechanism of retention.

The sensitivity of the me thod was investigated by preparing various cali- bration plots for several representative pesticides and aliphatic phosphorus compounds. In both cases, the calibration plots were prepared by replicate injection of a mixture of compounds that was completely resolved. For the pesticides, standards containing 0.1, 0.5, 1.0, 5.0, and 10.0 ~g were used. Plots of amount injected vs. peak height, in absorbance, were found to be linear (r = 0 .9991--0.9999) over the range studied. The detect ion limits, defined as three times the peak-to-peak noise, are tabulated together with the regression equations [ 14] in Table 4. The highest sensitivity was obtained with methyl parathion because this compound is aromatic and has a chromo- phore in the ultraviolet region. Phorate, being aliphatic, showed the lowest sensitivity. The sensitivity in the absence of the dye is lower for compounds having an absorbing chromophore, and is practically zero for compounds wi thout chromophores.

Calibration plots for o ther aliphatic compounds were prepared as men- t ioned before. Standards containing 70, 200, 300, 500, and 700 gg were injected. The calibration plots obtained (Table 4) are linear (r -- 0.976-- 0.998) over the concentrat ion ranges studied. The sensitivity is about the same for all three compounds because these compounds themselves do not absorb. Thus the response measured is exclusively due to the dye-analyte interaction.

The limit of detect ion for the aliphatic compounds are about one hundred times larger than the corresponding detection limits obtained with the aromatic compounds (Table 4). This difference is at t r ibuted to the contribu- tion of the absorbing chromophores in the aromatic compounds.

Even though the sensitivity obtained with the present method is modest , it is still comparable to that of other h.p.l.c, methods reported recently in which opt imum detect ion condit ions were used [6, 15] .

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TABLE 4

Calibration plots for organic phosphorus compounds a

Compound Slope (m) Intercept (b) Detection Std. -+ s.d. ± s.d. limit (ug) error

(x 10)

Pesticides b Ethyl 17.71 ± 0.20 1.36 ± 0.91 0.10 1.98 parathion Coumaphos 9.58 -+ 0.05 --0.21 -+ 0.23 0.20 0.50 Methyl 44.24 ± 0.64 4.88 ± 2.93 0.10 6.39 parathion Diazinon 6.83 -+ 0.17 1.16 + 0.84 0.20 1.67 Phorate 2.88 -+ 0.08 0.18 ± 0.19 0.50 0.76

Aliphat ic c o m p o u n d s c DEPI 8.11 ~ 0.28 3.64 + 1.28 37.0 0.03 HMP 7.40 ± 0,45 9.98 ± 1.90 40.0 0.04 DEEPO 5.28 ± 0.68 16.5 -+ 2.89 57.0 0.07

aMobile phase containing 0.10 mM dye, 2.0 mM ammonium phosphate and 50% (v/v) acetonitrile--water (for pesticides) or 30% (v/v) methanol--water (aliphatics). Detection at 254 nm. H is peak height in milliabsorbance; C is the amount of sample injected in ~g, and s.d. is the standard deviation, bRegression eqn.: H --- mC + b. CRegression eqn.: H = m C / l O 0 + b.

This research was supported by a grant from the Office of Naval Research.

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